The proposed research project focuses on some basic, unresolved aspects of the physiologically important role of thrombin as an anticoagulant. The project builds upon developments from previous NIH support and consists of the following specific aims: 1. elucidate the differential effect of Na+ on the cleavage of fibrinogen and protein C by thrombin;2. elucidate the role of W215 in the recognition of fibrinogen and protein C;and 3. solve the structures of wild-type and mutant thrombins in complex with thrombomodulin. We will use a combination of kinetic, site-directed mutagenesis and X-ray structural studies. In specific aim 1, we will address a basic unresolved issue of thrombin function, i.e., why Na+ promotes fibrinogen cleavage by thrombin but has no effect on protein C activation. Parallel mutagenesis studies on murine thrombin will test the hypothesis that its molecular mimicry of the Na+-bound form translates into resistance to mutations that perturb Na+ binding and stabilize the anticoagulant Na+-free form. These studies will produce a much needed understanding of the molecular basis of the Na+-dependent switch in specificity of human thrombin and of the molecular strategy utilized by the murine enzyme to optimize its procoagulant activity. In specific aim 2, we will carry out saturation mutagenesis of residue W215 and establish to what extent the specificity of thrombin can be altered in favor of fibrinogen or protein C. Mutations of W215 that produce significant anticoagulant effects will be combined with other mutations to produce a new generation of anticoagulant thrombin mutants that has completely lost activity toward fibrinogen, but retain activity toward protein C. These studies will deepen our understanding of how thrombin recognizes physiologic substrates at the active site and will produce new reagents of potential pharmacological relevance. In specific aim 3, we will solve the X-ray crystal structures of wild-type and mutant thrombins, human and murine, that are particularly relevant to the molecular basis of thrombin anticoagulant activity. We will give top priority to solution of the structure of wild-type and selected mutants in complex with thrombomodulin. Anticoagulant thrombin mutants will be crystallized free and bound to active site inhibitors. These studies will significantly broaden our current knowledge of how thrombin interacts with thrombomodulin at the molecular level, and how mutations of the enzyme result in the remarkable anticoagulant effects documented in vitro and in vivo studies.